Apr 22, 2025
C. neoformans Electroporation Transformation
This protocol is a draft, published without a DOI.
- Thomas Davies1
- 1University of Edinburgh
Protocol Citation: Thomas Davies 2025. C. neoformans Electroporation Transformation. protocols.io https://protocols.io/view/c-neoformans-electroporation-transformation-ehdebb23f
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 22, 2025
Last Modified: April 22, 2025
Protocol Integer ID: 136326
Abstract
Protocol for electroporation transformation of digested plasmid or PCR products into C. neoformans utilised in Hardwick lab. Adapted from "Modified Transient CRISPR-Cas9 Coupled with Electroporation(TRACE)", see references.
Materials
Buffers
- EB-Buffer: 10 mM Tris-HCl pH 7.5, 1 mM MgCl2, 270 mM Sucrose. Filter sterilised.
- 1M DTT
- Ice-cold ddH2O
Materials
- Electroporation cuvettes: We use Cell Projects, 2mm Gap (Cat. EP-102)
Preparation & Precultures
Preparation & Precultures
2-3 days prior to transformation, recover strains of interest from -80C stocks, patching to YPDA plates.
1 day prior to transformation, prepare precultures from plated strains, and grow at 30 °C o/n to OD600 0.2 on the morning of transformation. 100mL preculture provides enough biomass for 4-5 transformations.
Prepare selective medium plates in YPDA, typical concentrations of standard antibiotics shown in Table 1, below.
| Antibiotic | Concentration | |
| Geneticin (G418) | 100µg/mL | |
| Hygromycin B (HYG) | 300µg/mL | |
| Nourseothricin (NAT) | 100µg/mL |
Prepare DNA for transformation. For digested plasmid transformation 5-10µg DNA should be utilised, phenol-chloroform extraction ethanol precipitation recommended for cleanup, especially of large plasmids. Resuspended in 10µL ddH2O.
Competent Cell Preparation
Competent Cell Preparation
On morning of transformation, check preculture OD and monitor growth at 30 °C to OD600 0.3-0.36. Meanwhile pre-chill sterile ddH2O and EB-Buffer on ice.
Centrifuge culture at 3200 rpm, 4°C, 00:03:00 to pellet cells, wash twice with 35mL ice-cold ddH2O. Cells can be kept on ice for a few hours if required.
All further steps should be carried out with ice-cold buffers and On ice .
Centrifuge washed cells at 3200 rpm, 4°C, 00:03:00 and resuspend in 10mL EB-Buffer, adding 1mM DTT. Incubate On ice 01:00:00 .
Centrifuge cells at 3200 rpm, 4°C, 00:03:00 , remove supernatant and resuspend in 250µL EB-Buffer. Should have roughly 500µL of cell slurry total.
Electroporation Transformation
Electroporation Transformation
To DNA On ice , add 40µL cell slurry and mix. Transfer to pre-cooled electroporation cuvette, being careful not to introduce bubbles.
Electroporate cells at 1.7kV, 600Ω, 25µF, pulse once and immediately place back On ice .
Add 1mL YPDA to electroporated cells, and transfer to 1.5mL eppendorf tube. Incubate200 rpm, 30°C, 02:00:00 , allowing cells to recover and begin expressing antibiotic resistance genes.
Spin down cells 2000 rpm, Room temperature, 00:03:00 , remove majority of supernatant and resuspend in ~100µL remaining YPDA. Plate to antibiotic resistance plates, spreading with beads, and incubate 30 °C .
Growth and Screening
Growth and Screening
Check after 2 days (may take up to 3 days for colonies to appear), once colonies appear, pick and streak for single colonies onto antibiotic containing plates to ensure pure single colony strains.
Once colonies appear upon streaked plates, patch to YPDA and grow for downstream screening via PCR or western blot.
Protocol references
Fan, Y. and Lin, X. (2018) ‘Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex’, Genetics. Oxford University Press (OUP). Available at: https://doi.org/10.1534/genetics.117.300656.